Mechanism of Cell Membrane Targeting by Vibrio Cholerae Cytolysin

描述(由申请人提供)：霍乱弧菌溶细胞素(VCC)是一种攻击人类细胞的有效致孔毒素(PFT)，尽管这种毒素的具体目标和致病作用尚不清楚。了解致病毒力因子如何识别宿主膜是开发阻断其作用的治疗方法的重要第一步。这一知识也可能有助于针对患病细胞的药物的开发，例如癌症或病毒感染的细胞。长期目标是从结构和机制的角度阐明成孔毒素是如何识别、组装和破坏细胞膜的。这项应用的总体目标是了解VCC如何靶向细胞膜上的基序，包括碳水化合物和胆固醇分子，这是实现这一目标的下一步。VCC毒素含有两个结构域，其折叠类似于糖结合凝集素蛋白。在各种对人类健康有重要影响的VCC毒素同系物上发现了类似的碳水化合物结合结构域，包括创伤弧菌产生的毒素，创伤弧菌是致命食物中毒和败血症的原因之一。这个项目的中心假设是VCC以一种特定的和可逆的方式识别膜。这涉及到毒素分子上的结合部位，这些结合部位增加了膜表面毒素的局部浓度。这项拟议工作的基本原理是，VCC结合基序的结构和功能表征将提供对识别位点的贡献的明确理解 使毒素具有皮摩尔溶细胞活性。为了实现这一目标，三个 具体的目标是：1)确定VCC辅助结构域针对细胞膜上的碳水化合物配体；2)从结构上表征VCC辅助结构域与它们各自的配体之间的相互作用，以了解配体选择性的机制；以及3)研究VCC识别真核细胞膜中胆固醇的机制。在第一个目标下，将结合糖链筛选和生物物理特性来鉴定和定量VCC识别的碳水化合物配体。在第二个目标下，将使用X射线结晶学的高分辨率结构分析来了解配体选择性的性质。在第三个目标下，将分析假定的胆固醇结合部位对VCC识别易感细胞膜的贡献。这种方法是创新的，因为它结合了全面的结构分析和新的筛选技术，以回答有关生物功能的基本问题。这项拟议的研究具有重要意义，因为它将阐明这种强大的通道形成毒素如何利用受体以高亲和力组装在细胞膜上。这些信息将为针对特定细胞膜的毒素和药物治疗传染病和癌症的努力提供信息。 公共卫生相关性：拟议的研究与公共健康相关，因为PFTs，如VCC，参与病原体定植和宿主损害的过程。了解毒素是如何攻击细胞的，对于制定抗击传染病的战略至关重要。因此，这项拟议的研究与NIH通过支持人类疾病的病因和治疗研究来改善国民健康的使命相关。

DESCRIPTION (provided by applicant): Vibrio cholerae Cytolysin (VCC) is a potent pore-forming toxin (PFT) that attacks human cells, although the specific targets of the toxin and role in pathogenesis are unknown. Understanding how pathogenic virulence factors recognize host membranes is an important first step towards developing therapies to block their action. This knowledge may also benefit the development of drugs that target diseased cells, such as cancer or virally infected cells. The long-term goal is to elucidate how pore-forming toxins recognize, assemble on, and disrupt cellular membranes from a structural and mechanistic perspective. The overall objective of this application, which is the next step towards obtaining this goal, is t understand how VCC targets motifs on cell membranes, including carbohydrate and cholesterol molecules. The VCC toxin contains two structural domains with folds similar to sugar-binding lectin proteins. Similar carbohydrate-binding domains are found on a variety of VCC toxin homologs with important human health implications, including toxins produced by Vibrio vulnificus, a cause of deadly food poisoning and sepsis. The central hypothesis of this project is that VCC recognizes membranes in a specific and reversible manner. This involves binding sites on the toxin molecule that increase the local concentration of toxin on the surface of the membrane. The rationale for the proposed work is that structural and functional characterization of VCC binding motifs will provide a clear understanding of the contribution that recognition sites make towards the picomolar cytolytic activity of the toxin. In order to accomplish this goal, three specific aims will be pursued: 1) Identify the carbohydrate ligands that VCC accessory domains target on cell membranes; 2) Structurally characterize interactions between VCC accessory domains and their respective ligands to understand the mechanism of ligand selectivity; and 3) Investigate the mechanism by which VCC recognizes cholesterol in eukaryotic membranes. Under the first aim, a combination of glycan screening and biophysical characterization will be used to identify and quantify carbohydrate ligands recog nized by VCC. Under the second aim, high-resolution structural analysis by X-ray crystallography will be used to understand the nature of ligand selectivity. Under the third aim, putative cholesterol binding-sites will be ana lyzed fo their contribution towards the recognition of susceptible cell membranes by VCC. The approach is in novative because it combines a comprehensive structural analysis with new screening technologies to answer fundamental questions about biological function. The proposed research is significant, because it will illuminate how this potent channel-forming toxin utilizes receptorsto assemble on cell membranes with high-affinity. This information will inform efforts to target toxins and drugs to specific cell membranes in order to treat infectious diseases and cancer. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because PFTs, such as VCC, are involved in the process of pathogen colonization and host damage. Understanding how toxins attack cells is essential for developing strategies to combat infectious disease. Therefore, the proposed research is relevant to NIH's mission to improve the health of the Nation by supporting research in the causes and cure of human diseases.